Differentiation of vertebrate skeletal myoblasts into multinucleated myofibers is a multistage process that involves the coordinated activation of a cell-type specific transcriptional program and morphological changes that include elongation, alignment and cell-cell fusion. Transcriptional regulation is essential for these processes but the activity of myogenic transcription factors and the formation and growth of myofibers are controlled at the post-translational level by signal transduction pathways such as p38 MAP kinase and FAK. However, how the signaling pathways that stimulate the differentiation program are initiated and regulated is poorly understood. Illumination of such mechanisms is important both for understanding fundamental aspects of muscle development and identification of potential therapeutic targets for muscle disease and atrophy. Cdo is a multifunctional, cell surface co-receptor that is a component of a limited number of signaling pathways and which promotes myoblast differentiation in vivo and in vitro. Cdo forms cis complexes with the cell-cell adhesion molecule N-cadherin, and N-cadherin ligation promotes Cdo-dependent activation of p38 MAP kinase. Preliminary results identify Pak2 as a transducer of the signal between N-cadherin and p38. In addition, Cdo forms cis complexes with the netrin receptor neogenin, and this interaction also promotes myogenesis. Netrin activates FAK in a neogenin- and Cdo-dependent manner. Collectively, this work illuminates mechanisms of promyogenic signal transduction and offers fundamental insights into mechanisms of myoblast differentiation. The Specific Aims are: 1) to identify the role of Pak2 in skeletal muscle development and myoblast differentiation;2) to determine the role of netrin-3 and mechanisms of netrin/neogenin/Cdo signaling in myoblast differentiation;and 3) to define Cdo-containing cell surface complexes biochemically and identify where and when they occur. The successful completion of the proposed aims will provide molecular insight into the process of myogenic signal transduction by revealing roles for newly identified players in cell-cell contact- mediated signaling and through in-depth analysis of Cdo-containing complexes during differentiation. Such information will have a significant impact on understanding mechanisms of muscle development and, potentially, muscle disease. Furthermore, these studies will be of high value to other developmental contexts where cell-cell communication occurs and in pathological states where it goes awry. PUBLIC HEALTH RELEVANCE: Specific signal transduction pathways stimulate differentiation of skeletal muscle cells but how they are regulated is poorly understood. The proposed research will illuminate these mechanisms, providing insight into fundamental aspects of muscle development and identifying potential targets for the treatment of muscle disease and atrophy.